EP1162205B1 - Verfahren zur Herstellung eines Produktes mit einem hohen Gehalt an 2-O-Alpha-D-Glucopyranosyl-L-ascorbicsäure - Google Patents

Verfahren zur Herstellung eines Produktes mit einem hohen Gehalt an 2-O-Alpha-D-Glucopyranosyl-L-ascorbicsäure Download PDF

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EP1162205B1
EP1162205B1 EP01305024A EP01305024A EP1162205B1 EP 1162205 B1 EP1162205 B1 EP 1162205B1 EP 01305024 A EP01305024 A EP 01305024A EP 01305024 A EP01305024 A EP 01305024A EP 1162205 B1 EP1162205 B1 EP 1162205B1
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Prior art keywords
ascorbic acid
acid
saccharide
concentration
column
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EP1162205A2 (de
EP1162205A3 (de
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Hiroshi Yamasaki
Koichi Nishi
Toshio Miyake
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Hayashibara Seibutsu Kagaku Kenkyujo KK
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Hayashibara Seibutsu Kagaku Kenkyujo KK
Hayashibara Biochemical Laboratories Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the present invention relates to a novel process for producing a high-content product of 2-O- ⁇ -D-glucopyranosyl-L-ascorbic acid (hereinafter abbreviated as " ⁇ G-AA", unless specified otherwise), a stabilized L-ascorbic acid.
  • ⁇ G-AA 2-O- ⁇ -D-glucopyranosyl-L-ascorbic acid
  • ⁇ G-AA As disclosed in Japanese Patent Kokai No. 183,492/91, ⁇ G-AA is known to have the following satisfactory physicochemical properties:
  • ⁇ G-AA is now widely used in a cosmetic field mainly and is expected for its explorative use in other various fields such as food products, pharmaceuticals, feeds, pet foods, and industrial materials.
  • ⁇ G-AA As a representative example of industrial process for producing ⁇ G-AA is, for example, a process as disclosed in Japanese Patent Kokai No. 183,492/91.
  • the process comprises the steps of contacting a solution containing L-ascorbic acid and an ⁇ -glucosyl saccharide compound(s) with a saccharide-transferring enzyme or glucoamylase (EC 3.2.1.3) to form ⁇ G-AA to obtain a solution comprising ⁇ G-AA, intact L-ascorbic acid, ⁇ -glucosyl saccharide compound(s), and other saccharides produced from the ⁇ -glucosyl saccharide compound(s); filtering the resulting solution; removing minerals from the filtrate by subjecting the filtrate to column chromatography using a cation-exchange resin (H-form); subjecting the demineralized solution to column chromatography using an anion-exchange resin to adsorb ⁇ G-AA and L-ascorbic acid on the anion-exchange
  • Japanese Patent No 6228183 discloses a method for purifying a saccharide derivative of L-ascorbic acid. This method comprises treating a solution containing a derivative in which saccharides are bound to the 6-position of L-ascorbic acid synthesised by enzymatic reaction, microbial cell reaction or organic chemical reaction with a C1 - type strongly basic anion exchange resin.
  • the object of the present invention is to provide an industrial-scale production of high ⁇ G-AA content products with relatively-high quality and satisfactory processibility, production cost, and yield.
  • high ⁇ G-AA content product(s) as referred to in the present invention means high ⁇ G-AA content product(s) which contain(s) at least 80% (w/w) of ⁇ G-AA ("% (w/w)” may be abbreviated as "%”, hereinafter) , on a dry solid basis (d.s.b.), preferably, at least 90%, and which may have any form of a liquid, paste, solid or powder.
  • the present inventors continued studies on a simpler process for producing high ⁇ G-AA content products by contacting a solution containing ⁇ G-AA and L-ascorbic acid with an ion-exchange resin packed in a column.
  • the object can be attained by using an anion-exchange resin as an ion-exchange resin to be packed in a column; allowing ⁇ G-AA and L-ascorbic acid to adsorb on the anion resin; feeding to the column an aqueous solution, as an eluent, of an acid and/or a salt with a concentration of less than 0.5 N to fractionate into a fraction rich in ⁇ G-AA and a fraction rich in L-ascorbic acid; and collecting the former fraction.
  • the process for producing high ⁇ G-AA content product is characterized in that it comprises the steps of allowing a saccharide-transferring enzyme with or without glucoamylase to act on a solution containing L-ascorbic acid and an ⁇ -glucosyl saccharide compound(s) to obtain a solution containing ⁇ G-AA, L-ascorbic acid, and saccharides; filtering the solution; demineralizing the filtrate; contacting the demineralized solution, as a material solution, with an anion-exchange resin packed in a column to adsorb on the resin ⁇ G-AA and L-ascorbic acid; washing the resin with water to remove saccharides from the column; feeding an aqueous solution of an acid and/or a salt with a concentration of less than 0.5 N to fractionate a fraction rich in ⁇ G-AA and a fraction rich in L-ascorbic acid; and concentrating the former fraction to obtain a high ⁇ G-AA content
  • ⁇ G-AA, and L-ascorbic acid can be separated by column chromatography using an anion-exchange resin, resulting in a cancellation of the column chromatography using a strong-acid cation exchange resin which is inevitably used in a conventional preparation as shown in FIG. 1.
  • a strong-acid cation exchange resin which is inevitably used in a conventional preparation as shown in FIG. 1.
  • the material solutions used in the present invention are aqueous solutions which contain ⁇ G-AA, L-ascorbic acid, and a saccharide(s).
  • the process for producing the material solutions should not be restricted to specific ones, independently of enzymatic or synthetic methods, as long as they provide solutions containing ⁇ G-AA, L-ascorbic acid, and a saccharide(s).
  • a preventative example of such a process one comprising a step of allowing a saccharide-transferring enzyme with or without glucoamylase to act on a solution containing L-ascorbic acid and an ⁇ -glucosyl saccharide compound(s), e.g., the process disclosed in Japanese Patent Kokai No. 183,492/91.
  • the material solutions preferably used in the present invention have a solid concentration, usually, of 1-75% (w/v), preferably, 10-70% (w/v), more preferably, 20-60% (w/v), and most preferably, 30-40% (w/v). Since L-ascorbic acid is susceptible to decompose under alkaline conditions, the material solutions should preferably be set to acid pHs, usually, pHs less than 7, preferably, pHs of 1.0-6.5, more preferably, pHs of 1.5-6.0.
  • impurities such as anions, organic acids, and amino acids chat are coexisted in the material solutions should be removed or lowered as much as possible because of the use of anion-exchange resins (OH-form).
  • L-ascorbic acid as referred to in the present invention means usually free L-ascorbic acid and may include L-ascorbates such as alkaline metal salts and alkaline earth metal salts of L-ascorbic acid.
  • the L-ascorbic acid, used in the saccharide-transferring reaction of the present invention includes not only free L-ascorbic acid but L-ascorbates such as sodium L-ascorbate, calcium L-ascorbate, and mixtures thereof as long as they do not hinder the present invention.
  • ⁇ G-AA as referred to in the present invention means not only those in free acid form but salts thereof as long as they do not hinder the present invention.
  • ⁇ -glucosyl saccharide compound(s) as referred to in the present invention means those which can form ⁇ -glycosyl-L-ascorbic acid including ⁇ G-AA and which are composed of equimolar or more ⁇ -D-glucosyl residues bound to L-ascorbic acid via the action of a saccharide-transferring enzyme.
  • ⁇ -glucosyl saccharide compounds are maltooligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose; partial starch hydrolysates such as dextrins, cyclodextrins, and amyloses; and others such as liquefied starches, gelatinized starches, and soluble starches.
  • ⁇ G-AA it should preferably be selected ⁇ -glucosyl saccharide compounds suitable for the saccharide-transferring enzyme used.
  • ⁇ -glucosidase EC 3.2.1.20
  • maltooligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose
  • dextrins with a DE (dextrose equivalent) of about 50-60 and partial starch hydrolysates can be preferably used as ⁇ -glucosyl saccharide compounds.
  • cyclomaltodextrin glucanotransferase (EC 2.4.1.19) is used as a saccharide-transferring enzyme
  • cyclodextrins and partial starch hydrolysates such as gelatinized starches and dextrins with a DE from less than one to about 60 are preferably used.
  • ⁇ -amylase (EC 3.2.1.1) as a saccharide-transferring enzyme
  • partial starch hydrolysates such as gelatinized starches and dextrins with a DE from less than one to about 30 are preferably used.
  • the concentration (% (w/v)) of L-ascorbic acid used in the above saccharide-transferring enzymatic reaction is usually at least 1% (w/v), preferably, about 2-30% (w/v), while the preferable concentration of an ⁇ -glucosyl saccharide compound is usually about 0.5-30 folds of that of the L-ascorbic acid used.
  • any saccharide-transferring enzymes can be used in the present invention as long as they form ⁇ -glycosyl-L-ascorbic acid such as ⁇ G-AA in which at least one ⁇ -glucosyl residue is transferred to the alcohol group at C-2 of L-ascorbic acid without decomposing L-ascorbic acid when allowed to act on an aqueous solution containing L-ascorbic acid and an ⁇ -glucosyl saccharide compound(s).
  • the ⁇ -glucosidases usable as the saccharide-transferring enzyme in the present invention include those which are animal origins such as those from murine renes, rat intestinal mucosas, and small intestines of dogs and pigs; plant origins such as those from rice seeds and corn seeds; and bacterial origins such as those from fungi of the genera Mucor and Penicillium and yeasts of the genus Saccharomyces.
  • saccharide-transferring enzymes i.e., cyclomaltodextrin glucanotransferase
  • examples of other saccharide-transferring enzymes i.e., ⁇ -amylases are those of bacterial origins of the genus Bacillus.
  • saccharide-transferring enzymes should not necessarily be in a purified form, and those in a crude form can be also used in the present invention. However, such crude enzymes can be preferably purified by conventional methods prior to use.
  • commercialized saccharide-transferring enzymes can be used. In use, the above enzymes can be arbitrarily used in an immobilized form. Any pHs and temperatures for the saccharide-transferring enzymatic reaction can be used in the present invention as long as the saccharide-transferring enzymes act on L-ascorbic acid and ⁇ -glucosyl saccharide compounds to form ⁇ G-AA: Usually, it is selected from pHs of 3-9, preferably, pHs of 4-7, and temperatures of about 20°C to about 80°C.
  • the amount of the enzymes used and the enzymatic reaction time are closely related each other, the amount of enzyme is usually chosen from the above range so as to complete the enzymatic reaction within about 3-80 hours from an economical viewpoint.
  • the saccharide-transferring enzymes can be advantageously used in a batch-wise or in a continuous manner.
  • L-ascorbic acid is susceptible to decompose by the oxygen coexisted in the reaction system; it should preferably be kept in a reduced oxygen or non-oxygen conditions or reducing conditions, and if necessary, thiourea and sulfites can be preferably coexisted. Because L-ascorbic acid is susceptible to decompose by light, it should preferably be reacted under dark- or light shielded-conditions.
  • ⁇ -glycosyl-L-ascorbic acid containing ⁇ G-AA can be formed by coexisting microorganisms with saccharide-transferring ability in growth media with L-ascorbic acid and ⁇ -glucosyl saccharide compounds.
  • the ⁇ -glycosyl-L-ascorbic acid formed via the action of a saccharide-transferring enzyme is a compound composed of one or more ⁇ -D-glucosyl residues bound to the alcohol group at C-2 of L-ascorbic acid.
  • the number of ⁇ -D-glucosyl residues, that are bound via the ⁇ -1,4 linkage, is usually about two to seven. Examples of such ⁇ -glycosyl-L-ascorbic.
  • ⁇ G-AA 2-O- ⁇ -D-maltosyl-L-ascorbic acid, 2-O- ⁇ -D-maltotriosyl-L-ascorbic acid, 2-O- ⁇ -D-maltotetraosyl-L-ascorbic acid, 2-O- ⁇ -D-maltopentaosyl-L-ascorbic acid, 2-O- ⁇ -D-maltohexaosyl-L-ascorbic acid, and 2-O- ⁇ -D-maltoheptaosyl-L-ascorbic acid.
  • ⁇ -glycosyl-L-ascorbic acid is formed by using ⁇ -glucosidase, only ⁇ G-AA is usually formed.
  • ⁇ -glycosyl-L-ascorbic acids such as 2-O- ⁇ -D-maltosyl-L-ascorbic acid and 2-O- ⁇ -D-maltotriosyl-L-ascorbic acid are formed together with ⁇ G-AA.
  • ⁇ -glycosyl-L-ascorbic acids which have more ⁇ -D-glucosyl residues than those produced with ⁇ -glucosidase, can be usually formed together with ⁇ G-AA.
  • ⁇ -glycosyl-L-ascorbic acids composed of about one to seven ⁇ -D-glucosyl residues bound to L-ascorbic acid are formed, while in the case of using ⁇ -amylase, it tends to form lesser types of ⁇ -glycosyl-L-ascorbic acids.
  • saccharide-transferring enzyme is preferably first allowed to act on L-ascorbic acid and an ⁇ -glucosyl saccharide compound(s) to transfer equimolar or more ⁇ -D-glucosyl residues to L-ascorbic acid and to form a mixture of ⁇ -glycosyl-L-ascorbic acids and ⁇ G-AA, and then allowing glucoamylase to act on the resulting mixture to hydrolyze ⁇ -D-glucosyl residues, that are bound to the ⁇ -glycosyl-L-ascorbic acids other than ⁇ G-AA, and to form and accumulate ⁇ G-AA.
  • the glucoamylase used in the present invention includes those which are from microorganism, plant, and animal origins. Usually, commercialized glucoamylases from bacteria of the genera Aspergillus and Rhizopus can be advantageously used. In this case, ⁇ -amylase (EC 3.2.1.2) can be arbitrarily used along with such glucoamylases.
  • any one of those in the form of a gel, macro-reticular (MR-form), or macroporous structure as a mother structure can be used, and any one of strong- or weak-base styrene (styrenedivinylbenzene copolymer) and acrylic anion-exchange resins can be used as a basal material.
  • Any anion-exchange resins in a strong-alkaline (I and II types), neutral, or week-alkaline form can be used in the present invention as long as they have anion-absorbing ability.
  • anion exchangers now commercially available are “AMBERLITE IRA67”, “AMBERLITE IRA96SB”, “AMBERLITE IRA400”, “AMBERLITE IRA401B”, “AMBERLITE IRA402”, “AMBERLITE IRA402BL”, “AMBERLITE IRA410”, “AMBERLITE IRA411S”, “AMBERLITE IRA440B”, “AMBERLITE IRA458RF”, “AMBERLITE IRA473”, “AMBERLITE IRA478RF”, “AMBERLITE IRA900”, “AMBERLITE IRA904", “AMBERLITE IRA910CT”, “AMBERLITE IRA958”, “AMBERLITE XT5007", “AMBERLITE XT6050RF”, “AMBERLITE XE583”, and “AMBERLITE CG400”, which are all commercialized by Rohm & Haas Co., Pennsylvania, U.S.A.; and “DIAION SA10A”, “DIAION
  • anion exchangers "AMBERLITE IRA411S”, “DIAION WA30”, “AMBERLITE IRA478RF”, and “AMBERLITE IRA 910CT” can be preferably used in the present invention because of their relatively high absorbability and separability of ⁇ G-AA and L-ascorbic acid.
  • anion exchange resins those in the form of OH, Cl and CH 3 COO can be appropriately used.
  • those in the form of OH can be advantageously used in view of their absorbability.
  • the eluents used in the present invention include one or more acids such as hydrochloric acid, sulfuric acid, nitric acid, and citric acid, as well as aqueous solutions thereof; and one or more salts such as sodium chloride, potassium chloride, sodium sulfate, sodium nitrate, potassium nitrate, sodium citrate, and potassium citrate, as well as aqueous solutions thereof.
  • acids such as hydrochloric acid, sulfuric acid, nitric acid, and citric acid
  • salts such as sodium chloride, potassium chloride, sodium sulfate, sodium nitrate, potassium nitrate, sodium citrate, and potassium citrate, as well as aqueous solutions thereof.
  • aqueous salt solutions as eluents ⁇ G-AA contained in eluates, which had been desorbed and eluted from columns packed with anion exchangers, is generally in a salt form.
  • ⁇ G-AA can be demineralized and converted into a free acid form of ⁇ G-AA by using cation exchangers (H-form).
  • H-form cation exchangers
  • aqueous acid solutions as eluents ⁇ G-AA contained in eluates, which had been desorbed and eluted from columns packed with anion exchangers, is generally in a free acid form.
  • aqueous solutions used as eluents in the present invention aqueous solutions of hydrochloric acid can be advantageously used form economical viewpoint and easiness of regeneration of anion exchange resins.
  • concentration of aqueous solutions of acids or salts used as eluents should not necessarily be set to a constant level, and if necessary, it can be changed step-wisely or linearly as in a gradient elution manner.
  • the eluents once used in desorbing and eluting ⁇ G-AA and L-ascorbic acid can be recovered and reused to lower the production costs.
  • the material solutions used in the present invention usually contain ⁇ G-AA, L-ascorbic acid, and ⁇ -glucosyl saccharide compounds, as well as ⁇ -glucosyl saccharide compounds, D-glucose from the ⁇ -glucosyl saccharide compounds, saccharides such as hydrolyzates of the ⁇ -glucosyl saccharide compounds, and salts.
  • the material solutions are first filtered, then demineralized using cation exchange resins (H-form) with or without activated carbons.
  • the demineralized solutions are contacted with anion exchange resins packed in columns to absorb thereupon ⁇ G-AA and L-ascorbic acid, followed by washing the columns with water to remove saccharides, feeding to the columns aqueous solutions of acids and/or salts with a concentration of less than 0.5 N as eluents to fractionate into a fraction rich in ⁇ G-AA and a fraction rich in L-ascorbic acid, and collecting the former fraction.
  • the separated intact L-ascorbic acid and ⁇ -glucosyl saccharide compounds can be reused as materials for the next saccharide-transferring reaction from an economical viewpoint.
  • the column used in the present invention can be constructed by one or more columns, having an appropriate shape, size, and length, cascaded in series or connected in parallel. When used plural columns, they can be cascaded in series to give an appropriate column bed-depth.
  • the flow rate for feeding the material solutions to anion exchange resins is preferably SV (space velocity) 5 or lower, more preferably, SV 1-3.
  • the flow rate for feeding eluents to anion exchange resins is preferably SV 1 or lower, more preferably, SV 0.3-0.7.
  • the order of a fraction rich in ⁇ G-AA and a fraction rich in L-ascorbic acid eluted from columns can be changed by altering the types of anion exchange resins used, particularly, by varying the mother structures and functional groups of the anion exchange resins.
  • the temperatures during column chromatography are usually those in the range of 0-80°C, preferably, 10-40°C, and more preferably, 15-25°C, by taking into consideration the influence of temperature on the decomposition of ⁇ G-AA and L-ascorbic acid.
  • ⁇ G-AA When used aqueous solutions of acids and/or salts with concentrations of at least 0.5 N, ⁇ G-AA is promptly and easily desorbed from anion exchange resins and eluted from columns, while L-ascorbic acid is also desorbed and eluted together with ⁇ G-AA, resulting in their insufficient separation and in a difficulty of obtaining fractions rich in ⁇ G-AA with a relatively-high purity and yield.
  • aqueous solutions of acids and/or salts with a total concentration of less than 0.5 N, preferably, 0.1-0.45 N, and more preferably 0.2-0.3 N ⁇ G-AA and L-ascorbic acid are respectively desorbed and eluted from anion exchange resins packed in columns as their respective enriched fractions, resulting in an efficient separation and collection of their enriched fractions in a satisfactorily-high yield.
  • fractions rich in ⁇ G-AA and fractions rich in L-ascorbic acid can be yielded even with aqueous solutions of acids and/or salts with concentrations of less than 0.1 N, however, the desorption and elution time required for ⁇ G-AA and L-ascorbic acid from anion exchange resins tends to longer and the concentration of the resulting ⁇ G-AA tends to lower.
  • fractions rich in ⁇ G-AA After purified and concentrated into supersaturated solutions, fractions rich in ⁇ G-AA, which had been desorbed and eluted from anion exchange resins packed in columns, can be facilitated to form ⁇ G-AA crystals and to yield a high ⁇ G-AA content product in a crystalline form. While fractions rich in L-ascorbic acid can be reused as a material for the next enzymatic reaction to form ⁇ G-AA. Reuse of mixture fractions of incompletely separated ⁇ G-AA and L-ascorbic acid, as a material solution in the present invention, advantageously facilitates to yield high ⁇ G-AA content products and high L-ascorbic acid content products in a relatively-high yield.
  • the process for producing ⁇ G-AA of the present invention has satisfactorily advantages in processibility and production cost and makes it possible to produce high ⁇ G-AA content products with satisfactory high-quality and a purity of at least about 90%, on a dry solid basis (d.s.b.), in a yield of ⁇ G-AA of at least 85%, preferably, at least 90% to the ⁇ G-AA in the material solutions.
  • the high ⁇ G-AA content products thus obtained are stable high L-ascorbic acid content products which can be arbitrarily used as a vitamin C-enriching agent, stabilizer, quality improver, antioxidant, physiologically-active substance, and ultraviolet absorbent in a wide variety of fields of food products, cosmetics, pharmaceuticals, feeds, pet foods, industrial materials, etc.
  • dextrin Two parts by weight of dextrin (DE of about six) were dissolved by heating in six parts by weight of water, and under reducing conditions, the solution was admixed with one part by weight of L-ascorbic acid. The mixture was kept at pH 5.5 and 60°C and admixed with 400 units/g dextrin of cyclomaltodextrin glucanotransferase, commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and enzymatically reacted for 24 hours.
  • DE dextrin
  • the reaction mixture was filtered with a UF membrane to remove the remaining enzyme, and the filtrate was adjusted to 55°C and pH 5.0, admixed with 10 units/g dextrin of glucoamylase, commercialized by Seikagaku-Kogyo Co., Ltd., Tokyo, Japan, and enzymatically reacted for 24 hours.
  • the resulting reaction mixture was heated to inactivate the remaining enzyme and decolored and filtered with an activate charcoal, followed by concentrating the resulting filtrate to give a concentration of about 40%.
  • the solid composition of the concentrate was analyzed on high-performance liquid chromatography (HPLC) under the following conditions: "LC-6A”, an HPLC apparatus commercialized by Shimadzu Corporation, Tokyo, Japan; "STR COLUMN ODS-II", a column commercialized by Shimadzu Techno Research Co., Tokyo, Japan; 0.02M NaH 2 PO 4 -H 3 PO 4 (pH 2.0), an eluent; 0.5 ml/min, a flow rate; and "RI-8020", a differential refractometer commercialized by Tosho Corporation, Tokyo, Japan.
  • HPLC-6A an HPLC apparatus commercialized by Shimadzu Corporation, Tokyo, Japan
  • STR COLUMN ODS-II a column commercialized by Shimadzu Techno Research Co., Tokyo, Japan
  • 0.02M NaH 2 PO 4 -H 3 PO 4 pH 2.0
  • RI-8020 a differential refractometer commercialized by Tosho Corporation, Tokyo, Japan.
  • the solid composition of the concentrate contained about 31% of ⁇ G-AA, about 21% of L-ascorbic acid, about 31% of D-glucose, and about 17% of ⁇ -glucosyl saccharide compounds.
  • the above filtrate was demineralized by feeding at SV 2 to a column, having an inner diameter of 20 mm, a length of 350 mm, and an inner column temperature of 25°C, packed with 100 ml of "DIAION SK1B (H-form)", a strong-acid cation exchange resin commercialized by Mitsubishi Chemical Co., Tokyo, Japan, and kept at an inner column temperature of 25°C.
  • Three hundred and thirty gram aliquots of the resulting eluate with a concentration of 30% were respectively fed at SV 2 to seven columns, each having an inner diameter of 20 mm, a length of 650 mm, and an inner column temperature of 25°C, packed with 200 ml of "AMBERLITE IRA411S (OH-form)", an anion exchanger commercialized by Rohm & Haas Co., Pennsylvania, U.S.A. Thereafter, about 1,000 ml water was fed at SV 2 to each column to wash the anion exchanger to elute non-adsorbed components from each column.
  • ABERLITE IRA411S OH-form
  • FIGs. 3 and 4 respectively show the elution curves for the concentration distributions, obtained based on the results by plotting the solid concentration of each component in each eluate when 0.1 N or 0.5 N aqueous hydrochloric acid solution was used as an eluent.
  • the axis of ordinates shows the solid concentration (% (w/w)), d.s.b., of each component, in each eluate;
  • the axis of abscissas shows the elution volume (ml) from a column;
  • the symbols "- ⁇ -", "- ⁇ -", and "- ⁇ -” show ⁇ G-AA, L-ascorbic acid, and saccharides, respectively. From FIG.
  • FIG. 5 is an elution curve that shows the concentration distribution drawn, based on the analysis data, by plotting the solid concentrations of each component in each eluate.
  • the axis of ordinates shows the solid concentration (% (w/w)), d.s.b., of each component to the solid contents in each eluate; the axis of abscissas, elution volume (ml) from the column; the symbol "- ⁇ -", ⁇ G-AA; the symbol "- ⁇ -", L-ascorbic acid; and the symbol "- ⁇ -", saccharides.
  • the high ⁇ G-AA content product, obtained in this example contained ⁇ G-AA with a purity of about 93%, d.s.b., in a yield or about 92% to the content of ⁇ G-AA in the material solution.
  • FIG. 5 it was revealed that most of the saccharides were not absorbed on the anion exchanger but slightly adsorbed thereupon and eluted therefrom with the eluent.
  • dextrin Two parts by weights of dextrin (DE of about six) were dissolved by heating in six parts by weight of water, and under reducing conditions, the solution was then admixed with one part by weight of L-ascorbic acid and further admixed with 400 units/g dextrin of cyclomaltodextrin glucanotransferase, commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and enzymatically reacted at pH 5.5 and 60°C for 24 hours.
  • DE dextrin
  • the reaction mixture was filtered with a UF membrane to remove the enzyme, and the filtrate was adjusted to 55°C and pH 5.0, admixed with 10 units/g dextrin of glucoamylase, commercialized by Seikagaku-Kogyo Co., Ltd., Tokyo, Japan, and subjected to an enzymatic reaction for 24 hours.
  • the resulting reaction mixture was heated to inactivate the remaining enzyme, decolored, and filtered with an activated charcoal, and then the filtrate was concentrated to give a solid concentration of about 40%,
  • the analysis of the solid composition of the concentrate under the same HPLC conditions as used in the above experiment revealed that the solid composition contained about 31% of ⁇ G-AA, about 21% of L-ascorbic acid, about 31% of D-glucose, and about 17% of ⁇ -glucosyl saccharide compound.
  • FIG. 6 shows an elution curve for concentration distribution drawn by plotting the solid concentration of each component in each eluate.
  • Example 6 the elution order of ⁇ G-AA and L-ascorbic acid was reversed to that of Example 1 because of the use of a different anion exchanger from that of Example 1, and the separation of ⁇ G-AA and L-ascorbic acid was satisfactory, similarly as in Example 1, yielding in a high ⁇ G-AA content product by collecting a desired fraction rich in ⁇ G-AA.
  • FIG. 6 the elution order of ⁇ G-AA and L-ascorbic acid was reversed to that of Example 1 because of the use of a different anion exchanger from that of Example 1, and the separation of ⁇ G-AA and L-ascorbic acid was satisfactory, similarly as in Example 1, yielding in a high ⁇ G-AA content product by collecting a desired fraction rich in ⁇ G-AA.
  • the axis of ordinates shows the solid concentration (%(w/w)), of each component to the solid contents in each eluate; the axis of abscissas, elution volume (ml) from the column; the symbol "- ⁇ -", ⁇ G-AA; the symbol "- ⁇ -", L-ascorbic acid; and the symbol "- ⁇ -", saccharides.
  • ⁇ G-AA and L-ascorbic acid were satisfactorily desorbed and eluted from the anion exchanger, yielding in a high ⁇ G-AA content product by collecting a desired fraction rich in ⁇ G-AA.
  • the product contained ⁇ G-AA with a purity of about 93%, d.s.b., and had a yield of about 90% to the content of ⁇ G-AA in the material solution.
  • FIG. 6 it was revealed that most of the saccharides were not absorbed on the anion exchanger but slightly adsorbed thereupon and eluted therefrom with the eluent.
  • a high ⁇ G-AA content product was produced under the similar conditions as used in Example 2 except for using a column (400 mm in inner diameter, 1,000 mm in length) packed with "DIAION SK1B (H-form)", a strong-acid cation exchanger commercialized by Mitsubishi Chemical Co., Tokyo, Japan, for demineralization; a column (560 mm in inner diameter, 1,000 mm in length, 25°C of inner column temperature), packed with "AMBERLITE IRA910CT (OH-form)", an anion exchanger commercialized by Rohm & Haas Co., Pennsylvania, U.S.A., as an anion exchanger; and 0.3 N aqueous nitric acid solution as an eluent.
  • the high ⁇ G-AA content product had a liquid form, a purity of about 92%, and an ⁇ G-AA yield of about 90% to the content of ⁇ G-AA in the material solution.
  • the liquid high ⁇ G-AA content product was concentrated in vacuo into a solution with a solid concentration of about 76%, placed in a crystallizer, admixed with one percent, d.s.b., of ⁇ G-AA crystal as a seed, kept at 40°C, and then moderately cooled to 25°C over two days under gentle stirring conditions and separated by using a basket-type centrifuge. The resulting crystals were washed by spraying with a small amount of water, followed by collecting ⁇ G-AA in a crystalline form to obtain a high ⁇ G-AA content product with a purity of about 99%, d.s.b.
  • a high ⁇ G-AA content product was produced under the similar conditions as used in Example 2 except for using 0.45 N aqueous hydrochloric acid solution as an eluent. similarly as in Example 2, ⁇ G-AA and L-ascorbic acid were satisfactorily eluted from the anion exchanger packed in a column.
  • the high ⁇ G-AA content product, obtained in this example contained ⁇ G-AA with a purity of about 90%, d.s.b., and had an ⁇ G-AA yield of about 91% to the content of ⁇ G-AA in the material solution.
  • a high ⁇ G-AA content product was produced under the similar conditions as used in Example 2 except for using a column (800 mm in inner diameter, 1,600 mm in length) packed with "DIAION SK1B (H-form)", a strong-acid cation exchange resin commercialized by Mitsubishi Chemical Co., Tokyo, Japan, for demineralization; a column (1,200 mm in inner diameter, 1, 600 mm in length, 25°C of inner column temperature) packed with "AMBERLITE IRA478RF (OH-form)", an anion exchanger commercialized by Rohm & Haas Co., Pennsylvania, U.S.A., as an anion exchanger; and 0.2 N aqueous sodium chloride solution as an eluent.
  • DIION SK1B H-form
  • AMBERLITE IRA478RF OH-form
  • the high ⁇ G-AA content product was a sodium salt form of ⁇ G-AA ( ⁇ G-AA-Na), containing ⁇ G-AA-Na with a purity of about 90% and had an ⁇ G-AA-Na yield of about 90% by molar ratio to the content of ⁇ G-AA-Na in the material solution.
  • the present invention enables to produce a high ⁇ G-AA content product and a high L-ascorbic acid content product in a relatively-high yield by a process comprising the steps of: contacting a solution, as a material solution, containing ⁇ G-AA, L-ascorbic acid, and a saccharide (s), with an anion exchange resin packed in a column, to adsorb the ⁇ G-AA and the L-ascorbic acid on the anion exchanger; washing the exchanger with water to remove the saccharide(s) therefrom; feeding to the column an aqueous solution, as an eluent, of an acid and/or a salt with a concentration of less than 0.5 N to fractionate a fraction rich in ⁇ G-AA and a fraction rich in L-ascorbic acid; and collecting the former fraction.
  • both a column chromatography using a strong-acid cation exchanger, that is requisite for pre-column chromatography using an anion exchanger as used in conventional process for producing high ⁇ G-AA content products, and the concentration step requisite for a pretreatment for column chromatography using a cation exchanger can be omitted, and this simplifies the production process of ⁇ G-AA, lowers the production cost, and yields a high ⁇ G-AA content product in a relatively-high yield.
  • the process of the present invention needs no column chromatography using a strong-acid cation exchange resin, and this enables to increase the ⁇ G-AA yield from a level of about 75-80% as in conventional processes up to a level of about 85% or higher, preferably, 90% or higher to the ⁇ G-AA in a material solution.
  • the present invention enables to provide a process for producing high ⁇ G-AA content products with a satisfactory processibility, production cost, yield, and quality.
  • the high ⁇ G-AA content products can be arbitrarily used as a stable high L-ascorbic acid content product, vitamin C-enriching agent, stabilizer, quality improver, anti-oxidant, physiologically active substance, ultraviolet absorbent, etc., in a variety of fields of food products, cosmetics, pharmaceuticals, feeds, pet foods, industrial materials, etc.

Claims (8)

  1. Verfahren zur Herstellung eines Produkts mit einem hohen Gehalt an 2-O-α-D-Glucopyranosyl-L-ascorbinsäure, das folgende Schritte umfasst:
    Inberührungbringen einer Lösung, die 2-O-α-D-Glucopyranosyl-L-ascorbinsäure, L-Ascorbinsäure und ein oder mehrere Saccharide enthält, als Materiallösung mit einem Anionenaustauscherharz in OH--Form oder CH3COO--Form, das zur Adsorption der 2-O-α-D-Glucopyranosyl-L-ascorbinsäure und der L-Ascorbinsäure an das Anionenaustauscherharz in eine Säule gepackt ist;
    Auswaschen des Anionenaustauscherharzes mit Wasser zum Entfernen des/der Saccharids/e aus dem Anionenaustauscherharz;
    Einbringen einer wässrigen Lösung einer Säure und/oder eines Salzes mit einer Konzentration von weniger als 0,5 N als Laufmittel in die Säule zur Fraktionierung einer an 2-O-α-D-Glucopyranosyl-L-ascorbinsäure reichen Fraktion und einer an L-Ascorbinsäure reichen Fraktion ; und
    Sammeln der an 2-O-α-D-Glucopyranosyl-Lascorbinsäure reichen Fraktion.
  2. Verfahren nach Anspruch 1, wobei die Materiallösung durch Einwirkenlassen einer Saccharid-Transferase mit oder ohne Glucoamylase auf eine Lösung mit L-Ascorbinsäure und einer α-Glucosylsaccharid-Verbindung erhalten wird.
  3. Verfahren nach Anspruch 1, wobei die Materiallösung durch Inberührungbringen mit einem Kationenaustauscherharz in H-Form zum Entfernen von Mineralstoffen erhalten wird.
  4. Verfahren nach Anspruch 1, wobei die Säure einen oder mehrere Bestandteile ausgewählt aus der Gruppe bestehend aus. Salzsäure, Schwefelsäure, Salpetersäure und Zitronensäure darstellt.
  5. Verfahren nach Anspruch 1, wobei das Salz einen oder mehrere Bestandteile ausgewählt aus der Gruppe bestehend aus Natriumchlorid, Kaliumchlorid, Natriumsulfat, Natriumnitrat, Kaliumnitrat, Natriumcitrat und Kaliumcitrat darstellt.
  6. Verfahren nach Anspruch 1, wobei die wässrige Lösung der Säure und/oder des Salzes eine Konzentration von 0,1 - 0,45 N aufweist.
  7. Verfahren nach Anspruch 6, wobei die wässrige Lösung der Säure und/oder des Salzes eine Konzentration von 0,2 - 0,3 N aufweist.
  8. Verfahren nach Anspruch 1, das weiterhin die Schritte der Konzentrierung und der Kristallisierung umfasst.
EP01305024A 2000-06-08 2001-06-08 Verfahren zur Herstellung eines Produktes mit einem hohen Gehalt an 2-O-Alpha-D-Glucopyranosyl-L-ascorbicsäure Expired - Lifetime EP1162205B1 (de)

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KR20080112210A (ko) * 2006-02-22 2008-12-24 가부시끼가이샤 하야시바라 세이부쓰 가가꾸 겐꾸조 휘발성 황화물류의 생성 억제제와 이것을 이용하는 휘발성 황화물류의 생성 억제 방법
WO2011027790A1 (ja) * 2009-09-03 2011-03-10 株式会社林原生物化学研究所 ２－Ｏ－α－Ｄ－グルコシル－Ｌ－アスコルビン酸無水結晶含有粉末とその製造方法並びに用途
US20110172180A1 (en) 2010-01-13 2011-07-14 Allergan Industrie. Sas Heat stable hyaluronic acid compositions for dermatological use
US9114188B2 (en) 2010-01-13 2015-08-25 Allergan, Industrie, S.A.S. Stable hydrogel compositions including additives
JP5056918B2 (ja) * 2010-07-20 2012-10-24 富士ゼロックス株式会社 用紙処理装置および画像形成装置
US9149422B2 (en) 2011-06-03 2015-10-06 Allergan, Inc. Dermal filler compositions including antioxidants
WO2014103475A1 (ja) 2012-12-27 2014-07-03 株式会社林原 アンチエイジング用皮膚外用組成物及びその製造方法
CN103755756B (zh) * 2014-02-11 2015-07-08 江苏诚信药业有限公司 一种制备维生素c葡萄糖苷的工艺系统及其工艺方法
CN103923136B (zh) * 2014-04-20 2019-01-18 厦门世达膜科技有限公司 一种抗坏血酸葡糖苷的生产方法
CN105461768B (zh) * 2014-08-25 2018-10-30 上海医药工业研究院 一种2-O-α-D-葡萄糖基-L-抗坏血酸的制备方法
WO2018021542A1 (ja) * 2016-07-29 2018-02-01 カーリットホールディングス株式会社 ２－Ｏ－α－Ｄ－グリコシル－Ｌ－アスコルビン酸金属塩、その酸化防止剤としての用途及びその粉末の製造方法

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DE60100642T2 (de) 2004-06-17

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